1. Field of the Invention
Embodiments of the present invention generally relate to gel electrophoretic immunoassay, and more specifically to on-chip electrokinetic methods for performing immunoassays. These immunoassays are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform for the immunoassays enables integration, parallel assays, automation and development of portable devices.
2. State of the Art
There is a great demand for methods and devices for rapid detection of molecules of interest in applications such as medical diagnostics, environmental monitoring, biological defense and pharmaceutical research. For example, in medical diagnostics, detection and quantitation of biomarkers (proteins indicative of a disease state) in bodily fluids form the basis of diagnosis and treatment of many diseases such as cancer and HIV. Immunoassays are one of the most widely used and sensitive techniques for detection and quantitation of analytes such as viruses, peptides, polynucleotides, proteins such as toxins, antibodies, and cytokines, and other small molecules. Immunoassays are based on specific recognition and binding of a biological ligand to another molecule, the prominent example being binding of an “receptor” molecule to an analyte such as an antigen, where the receptor molecule may be any species having a specific binding affinity for another species. Reporter molecules may include but are not limited to polyclonal or monoclonal antibodies; a Fab, F(ab′) 2, scFV, or small chain fragment; a peptide or a peptide nucleic acid; an aptamer; lectin; one or more small ligands; an antigen; an enzyme; an oligonucleotide; a deoxyribonucleic acid; a ribonucleic acid; biotin; and cellular receptor binding proteins. The generality of immunoassays stems from the fact that most analytes implicated in disease progression are either antigens or antibodies or are molecules against which an antibody can be generated by utilizing the immune system of a host animal. Typically either the antibody or the antigen, or in many cases a secondary antibody, is labeled with a signal-generating molecule, or “reporter” molecule, such as a fluorescent molecule, a chemiluminescent molecule, an enzyme, a quantum dot, biotin, or a spin-label, to transduce the binding event into a measurable signal.
A typical immunoassay, such as in Enzyme-Linked Immunosorbent Assay (ELISA), is performed using a solid surface to immobilize one of the binding components (antibody or antigen). Multiple subsequent incubations and washing steps are required to separate the bound from unbound species—allowing for detection of only those species that have bound. In a sandwich ELISA using a microtiter plate, the antibody to the antigen of interest is adsorbed to a solid surface, in this example the bottom of the microtiter plate well. The surface is then blocked to eliminate nonspecific binding in subsequent steps by adsorbing a protein such as bovine serum albumin, followed by aspiration and rinsing to remove the unbound protein. In the second step, samples containing the antigen are incubated with the solid surface and the nonspecifically bound antigen is removed by washing. In the third step, a second antibody, also specific to the antigen, which is conjugated to one enzyme or fluorescent reporter molecule, is added. The amount of labeled antibody and, hence, the antigen is determined by assaying for the enzyme or detection of a fluorescent signal.
There exist numerous variations of immunoassays. ELISA, as described above, requires separation or washing steps, the ELISA is classified as a heterogeneous immunoassay. Whereas homogeneous immunoassays are performed where there is no need to perform separation or washing before quantitation—a signal is generated only from the bound analyte-antibody complex.
The conventional immunoassays, ELISA being the predominant variation, take a long time (hours) to complete as there are multiple incubation and washing steps involved. These assays also involve many steps that either require extensive labor or if automated, need large and expensive robotic liquid-handling equipment.
There has been extensive commercial and research interest in developing immunoassays that are fast, can be performed in portable devices and require minute amounts of sample and reagents.
Microchip Analysis
Microfluidic chips for analysis of biological molecules have attracted significant attention recently as they offer a number of advantages including speed of analysis, portability, ability to multiplex, and potential for integration.
Microfluidic systems employ microfabrication technologies borrowed from the microelectronics industry to form a network of microchannels (1 μm-200 μm in width and depth) in common materials such as glass or plastic. Many biochemical processes such as mixing, dilution, concentration, transport, separation, and reaction can be integrated and automated in a single chip. The ability to make multiple channels, without additional cost or time of fabrication, enables as many as 96 or more analyses to be performed simultaneously. Another key advantage offered by these systems is that they require, and are capable of handling, a very small amount of sample and reagent for each process—a few tens or hundreds of nanoliters, volumes that are impossible to analyze in conventional microtiter plates or vials as they will evaporate in seconds and are nearly impossible to handle.
In the last few years, routine biochemical methods have been adapted to microfluidic platforms without loss in performance. In fact, in many instances, miniaturization improves the performance in addition to enabling high throughput operation using vanishing small amounts of a biological sample. Methods such as protein and DNA electrophoresis, chromatography, cell sorting, and affinity assays (e.g., immunoassays) have been adapted to microchips. The microfluidic assays are typically faster, use 100-1000 times lower sample and reagents, and offer better separation resolution and efficiency than their conventional counterparts.
Another advantage that miniaturization offers is the ability to integrate different biochemical processes and components required to perform them. A complex microfluidic chip contains multiple liquid reservoirs, fluid channels, and materials to perform such diverse tasks as filtering, pumping, valving, dialysis, separation, detection, and the like. Compared to larger fluid-handling and analysis systems, an integrated microfluidic chip performs these tasks much faster using smaller amounts of reagents and has the potential to be significantly cheaper if mass produced. Integrating functions at microscale also greatly reduce sample loss and dilution, potentially allowing detection of amounts not possible at larger scale operation.
Electrophoretic Immunoassays
As explained earlier, a typical immunoassay is performed using a solid surface to immobilize one of the components (antibody or antigen) with multiple subsequent incubations and washing steps to separate the bound from unbound species. However, conventional assay methods generally require long incubation periods (hours) and appreciable amounts of sample and reagents in order to obtain the desired response. Electrophoresis in microchannels has been demonstrated as an efficient means to separate an immune complex from reporter molecules. In such systems, an immune complex and a reporter molecule, such as a fluorophore, are separated based upon differences in charge-to-mass ratios. Specific advantages of microdevice-based separations relevant to electrophoretic-based immunoassays include the potential for shortened incubation times (as compared to solid-phase systems), simplified assay protocols as compared to the multiple wash and detection steps required for conventional immunodiagnostics such as ELISA, and device form-factors amenable to system integration and automation. Additionally, electrophoretic immunoassays eliminate the need to immobilize analyte on a solid surface, thus avoiding complexities associated with the solid-phase. Several groups have demonstrated microdevices as an elegant architecture for conducting integrated immunoassays (see for instances Chiem, N., et al., Anal. Chem. 1997, v. 69, pp. 373-378; Koutny, L. B., et al., Anal. Chem. 1996, v. 68, pp. 18-22; Qiu, C. X., et al., Electrophoresis 2001, v. 22, pp. 3949-3958; and Cheng, S. B., et al., Anal. Chem. 2001, v. 73, pp. 1472-1479).
Polyacrylamide Gel Electrophoresis (PAGE) Immunoassays on Chips: Advantage of Cross-Linked Gels—Integration, Higher Sample Loading
To date, the capillary- or microchip-based immunoassays are performed in an open or surface-modified microfluidic channel and predominantly use electrophoresis as the basis of separation. The open-channel assays suffer from a number of disadvantages: 1) Attaining adequate species discrimination with electrophoresis-based immunoassays, however, can be difficult since large analytes such as antibodies and immune complexes are known to vary little in charge-to-mass characteristics. 2) Open channel electrophoresis also suffers from non-specific adsorption. Antibodies or analytes can adsorb to the walls leading to loss of sample as well as degradation in assay performance as the adsorbed molecules can significantly alter the flow in the channels. Various surface coatings, both covalent and non-covalent, have been developed to reduce non-specific adsorption but these coatings are not stable and lead to irreproducible results. Moreover, in most cases these coatings reduce but do not eliminate non-specific adsorption—leading to significant changes in the flow profile and hence, assay performance and reproducibility.
PAGE-electrophoresis through a porous sieving polyacrylamide, in the forms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE, is a widely-used method for separation of proteins. At conventional scale, PAGE is performed in cross-linked polyacrylamide gels sandwiched between glass plates. Conventional PAGE takes hours to run, needs large amounts of sample and is hard to automate and integrate. As explained in commonly owned U.S. patent application Ser. No. 10/646,808 entitled “Multidimensional Electrophoresis and Methods of Making and Using Thereof”, originally filed Aug. 25, 2003, and which is herein incorporated by reference, we have recently developed methods to implement slab gel electrophoresis in microchips. The major challenge in performing PAGE in a chip is the difficulty of placing solid cross-linked gels in micron-sized channels.
However, we have overcome these difficulties and herein describe and have elsewhere reported (Herr, A. E. et al., “On-chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin,” Anal. Chem.; v. 77(2), Jan. 15, 2005: pp. 585-590, and Herr, A. E. et al., “Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing,” Anal. Chem.; v. 76(16), Aug. 15, 2004: pp. 4727-4733, both herein incorporated by reference) a new method for providing a polyacrylamide gel in a microchannel and conducting separation assays for a variety of proteins including TTC (tetanus toxin C-fragment), IL-2 (recombinant human interleukin-2), FGF (recombinant human fibroblast growth factor), and IGF (recombinant human growth factor-I).
By using ultra-violet (UV) light to initiate polymerization, porous polymers can be formed in the channels of a microchip. Moreover, because polymerization is initiated by UV-light, the channels can be photolithographically patterned. Using a mask, the polymerization is restricted to UV-exposed regions, and monomers from the unexposed regions are flushed after the irradiation step. This allows polymer to be cast selectively in separation channels, while injection channels and the detection window remain open. This allows for rapid and repeatable injection, easy clean up of injection arms, and more sensitive detection. The ability to photopattern will also facilitate multi-dimensional separation by enabling multiple separate stationary phases in a single chip. Electrolytes are incorporated into the monomer mix allowing for generation of electrokinetic flow immediately after polymerization. This obviates need of pumps to condition the channels by removal of excess solvent and monomers.
SDS-PAGE allows for excellent discrimination of species by size, but sodium dodecyl sulfate can disrupt fragile immune complexes, making quantization of these complexes nearly impossible. Non-denaturing PAGE techniques, both with and without a detergent, have been shown to retain the biological activity necessary for intact immune complexes, yet allow analyte discrimination. The ability to discriminate between antigen, antibody, and immune complexes based upon size, as well as charge-to-mass ratio, mitigates the sometimes poor resolution observed using non-sieving electrophoretic immunoassay techniques. Our PAGE immunoassay is superior to zone electrophoretic immunoassays in many respects: 1) high separation resolution due to low non-specific binding, 2) fast separations (seconds) using short length channels (millimeters) due to the high surface area of the gel, 3) ready tailoring of gel porosity for specific applications, and 4) spatial-localization of photopatterned polyacrylamide (useful in multiplexing and integration).